Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

Transcription

1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Chan KCA, Woo JKS, King A, et al. Analysis of plasma Epstein Barr virus DNA to screen for nasopharyngeal cancer. N Engl J Med 2017;377: DOI: /NEJMoa

2 This supplement contains the following items: 1. final research protocol, original research protocol, and summary of changes; 2. final statistical plan, original statistical plan, and summary of changes.

3 Final Research Protocol Title: Prospective 20,000-person nasopharyngeal carcinoma screening programme using plasma Epstein-Barr virus (EBV) DNA analysis Principal investigator: - Professor KC Allen Chan Co-investigators: - Professor YM Dennis Lo - Professor Andrew C van Hasselt - Dr. John KS Woo - Professor Benny Zee - Professor Anthony Chan - Dr. SF Leung Objectives: To investigate if an NPC screening program using plasma EBV DNA would result in the detection of NPC at an earlier stage when compared with the nonscreened population To investigate if subjects with positive plasma EBV DNA would have an increased risk of future NPC development To determine the optimal frequency for NPC screening Background: Nasopharyngeal carcinoma (NPC) is one of the commonest cancers in Southern China and Southeast Asia. 1 In contrast to most other cancers which occur more commonly in elderly subjects, NPC is most prevalent in the middle age. The yearly incidence of NPC in men of age 40 to 60 in Hong Kong is as high as 40 cases per 100, The most important prognostic factor for NPC is the extent of disease involvement at presentation. In patients with localized disease, the 5-year survival probability is up to 90%. 2 This figure drops to below 50% in patients with metastatic disease. In addition, patients with more advanced disease would suffer from a lot more treatment-associated morbidities. 2 Unfortunately, 75% - 90% of NPC patients have already developed local or regional spread at presentation. 2,3 Therefore, screening for early disease in asymptomatic individuals may potentially improve the treatment outcomes of NPC. Epstein-Barr virus infection is an important aetiological factor for NPC. In Asia, EBV genome is detectable in almost all NPC tumour tissues. 4 Making use of the close relationship between EBV infection and NPC, our group has pioneered the development plasma EBV DNA as a tumour marker for NPC in 1999 with the funding support from the Kadoorie Charitable Foundation. 5-9 We showed that EBV DNA is detectable in the plasma of 96% NPC patients using real-time polymerase chain reaction (PCR). In contrast, only 2-7% of individuals without any evidence of NPC would have detectable plasma EBV DNA and at much lower levels than NPC patients. 5,10 The EBV DNA analysis was shown to be much superior to the existing standard surrogate marker for

4 NPC, the EBV IgA serological markers, in terms of sensitivity and specificity for detecting established NPC. 11 For the clinical management of NPC patients, plasma EBV DNA analysis is particularly useful for identifying clinically concealed residual NPC after curative intent radiotherapy 6,12 and for predicting treatment outcomes. 7,13 The prognostic value of circulating EBV DNA analysis has further been shown to be independent of the clinical stage of the NPC patients. 3 The clinical value of circulating EBV DNA analysis has been independently confirmed by other research groups Therefore, it has been proposed that plasma EBV DNA analysis could usefully be incorporated into the staging system of NPC. 3 We further carried out a prospective study to investigate the feasibility of using plasma EBV DNA analysis for the screening of early NPC in high risk asymptomatic individuals from 2007 to Over 1,300 volunteers of age 40 to 60 were recruited for the study. Three plasma EBV DNA analyses were performed for each participant at enrolment, 6 months and 18 months. Serological analysis for EBV viral capsid antigen (VCA) IgA was also performed at enrolment. After each screening session, endoscopic examinations of the nasopharynx were performed within four weeks for all individuals with positive test results (either EBV DNA or EBV VCA-IgA). Another blood sample was collected from each of them on the day of endoscopic examination for plasma EBV DNA testing to investigate the short-term variation of plasma EBV DNA concentrations. Three NPC cases were identified during the screening session at enrolment. All of the three NPC cases were positive for plasma EBV DNA but only one was positive for EBV VCA-IgA indicating that plasma EBV DNA is more sensitive than the serology test for identifying early NPC. In the three patients identified, two of them were completely asymptomatic. The third patient noted a solitary lymph node three months before joining the study. However, fine needle aspiration of the lymph node only showed benign reactive changes but no evidence of malignancy. Remarkably, the tumour of this patient was not detectable during the initial nasal endoscopic examination. Subsequently, prompted by the doubling of the plasma EBV DNA concentration taken two weeks later, a magnetic resonance imaging (MRI) was performed and showed an infiltrating tumour at the nasopharynx. The diagnosis of NPC was then confirmed by repeating the endoscopic examination and taking blind biopsies of the nasopharynx. These results indicate that plasma EBV DNA is useful for the screening of early NPC in apparently healthy individuals. Without this screening programme, these three patients are likely to present with more advanced disease when the symptoms become more overt. In addition to screening for early NPC, this previous study has also provided important information regarding the short- and intermediate-term of variation for plasma EBV DNA in subjects without an NPC. In each of the three screening sessions, 5% to 9% of the participants had detectable levels of EBV DNA in plasma. After a period of two to four weeks, two-thirds of the individuals with initial positive results would become negative for plasma EBV DNA, indicating that the presence of EBV DNA in plasma is only a transient event in a large proportion of subjects without an NPC. On the other hand, EBV DNA is persistently detectable in the plasma of the three NPC patients before treatment. These results suggest that repeating plasma EBV DNA analysis in two weeks for subjects with initial positive results would be useful for cutting down the proportion of subjects requiring nasal endoscopy for confirming or ruling out NPC. This

5 arrangement would significantly enhance the cost-effectiveness of the screening programme. Although the presence of plasma EBV DNA in a large proportion of non- NPC subjects was transient in the short term, subjects with detectable EBV DNA in plasma were significantly more likely to become positive again in the subsequent analyses carried out in 6 months and 18 months. During the previous study, approximately 9% of the individuals had detectable plasma EBV DNA in the two screening sessions carried out in winter time and only around 5% of the subjects had positive results in the session carried out in summer. There was a significant difference in positive rates between the sessions carried out in winter and summer. Using ultracentrifugation analysis, we showed that the plasma EBV DNA in non-cancer subjects are likely to be associated with intact virions instead of existing as free DNA fragments as in the NPC patients. The apparent seasonal variation could be the result from the more active viral replication in winter. If this observation is confirmed with long term studies, it would imply that NPC screening would best be performed in summer to minimize the false-positive rate. Questions to be answered in the current study Although all the three identified patients had early-staged diseases (Stages I and II) in the previous study, it does not have adequate statistical power to show that NPC screening could result in a down-staging of the NPC cases at diagnosis when compared with the non-screened population due to the relatively small size of the cohort. In the currently proposed study, we plan to use a 20,000-person cohort to establish the value of an NPC screening with regard to the down-staging of the identified NPC cases. Furthermore, we aim to determine the optimal frequency for NPC screening by determining the interval between the emergences of new NPC cases after those subjects being screened negative. In the previous study, we have shown that subjects had detectable plasma EBV DNA at one time point were more likely to be positive again subsequently. The biological significance of having recurrently or persistently detectable plasma EBV DNA remains unclear. In this regard, a previous study showed that subjects with a positive EBV serology have a 4- to 10-fold increased risk of developing NPC. 20 As EBV infection is an important aetiological factor for NPC, it is possible that the presence of circulating EBV DNA and EBV antibodies may reflect the pathological processes underlying NPC development. In the currently proposed study, the cohorts of subjects being positive and negative for plasma EBV DNA at recruitment will be followed up to investigate if the presence of detectable EBV DNA in plasma is associated with an increased risk of future NPC development. Study design: Subjects 20,000 Chinese male subjects of age 40 to 62 years will be recruited for the module 1 study over a period of two years. This target can be accomplished by recruiting approximately 250 subjects per week (250 subjects/week x 40 weeks/year x 2 years = 20,000 subjects). As the incidence of NPC in male is higher than in females, this study will focus on the male subjects of age 40 to 62 so as to maximize the chance of

6 identifying new NPC cases. The incidence of NPC would drop for older age groups. Subjects with an existing cancer or a past history of cancer, immunodeficiency or autoimmune diseases will be excluded. Subjects who are on immunosuppressive therapies, e.g., following transplantation, will also be excluded. In the previous proof-of-principle study, approximately 1,300 volunteers were recruited via 12 community visits. Through that exercise, we have established close working relationship with the district councillors and this is essential for the recruitment of volunteers for the currently proposed study. As the public awareness of the prevalence of NPC in Hong Kong is high, the public is generally interested in the voluntary participation in NPC screening programme. In addition to recruiting via community visits with the district councillors, we would invite large corporations in Hong Kong for collaborations for the recruitment of their staff to participate in this NPC screening exercise. Examples of these corporations include the Hospital Authority of Hong Kong, the universities, the banks, the two electricity companies and the public transportation companies. Webpages will be setup for the booking and arrangement of the screening sessions. Plasma EBV DNA analysis Plasma EBV DNA analysis will be performed for each participant during the first visit. An interview by the research nurses will be carried out to document the personal details, and the personal and family medical history relevant to NPC. Quantitative measurement of plasma EBV DNA will be performed by real-time PCR targeting the BamHI-W fragment of the EBV genome as previously described. 5 All samples will be analysed in duplicates. Any detectable signal regardless of the level in any of the duplicates will indicate a positive sample. Plasma EBV DNA analysis will be repeated for all subjects with an initial positive result in approximately four weeks. In the previous study, we have demonstrated that NPC patients would have persistently positive results before treatment but two-thirds of the subjects with false-positive results would have undetectable plasma EBV DNA on the follow-up analysis. Therefore, in the currently proposed study, only subjects with persistently detectable plasma EBV DNA at baseline and at the follow-up (defined as screen-positive) will be referred to an otorhinolaryngologist for further assessment. This arrangement could enhance the cost-effectiveness of the screening exercise. A MRI will also be performed for all screen-positive subjects. This arrangement would maximize the power for detecting NPC so as to provide the best ascertainment of the performance of plasma EBV DNA screening. In our previous studies, VCA-IgA has been shown to have inferior sensitivity over plasma EBV DNA for the detection of NPC, in particular, for early staged disease. 11 In the screening study that we have just completed, all three identified NPC patients had detectable plasma EBV DNA but only one of them was positive for VCA-IgA. The EBV serology test does not appear to provide addition value for the detection of early NPC and, hence, the testing for EBV serology will not be performed for the proposed study. Questionairre

7 Demongraphic and relevant clinical information will be collected from each participants. The information collected is as follows: Name Id number Sex Date of birth Age Occupation Address Phone number (of the subject and next of keen) Smoking (and details) Drink (and details) Exercise habit (hours of exercise per week and strength) Personal history of NPC and other cancers Family history of NPC History of organ transplantation HIV infection History of autoimmune disease Drug history (in particular immunosuppressants and systemic steroid) History of co-morbidities ENT symptoms including o epistaxis o enlarging neck lumps o nose block (unilateral or bilateral) o post-nasal drip o blood-stained nasal discharge of sputum o hearing loss (unilateral or bilateral) Follow-up After the initial NPC screening session, all the participants will be phoned and interviewed on a yearly basis to update their health records for two to three years. The development of NPC or other cancers will be documented. The interval between NPC development from the time of the plasma EBV DNA screening in patients who subsequently developed NPC in the follow-up period would be useful for determining the optimal frequency of carrying out NPC screening. After the two-year follow-up period, the list of the participants will be matched with the NPC list in the Hong Kong cancer registry yearly to identify new NPC in that year. The follow-up period of these cases is expected to be up to around 10 years. Then, the potential association between the presence of detectable EBV DNA in plasma and the risk of NPC development can also be analyzed. Following information will be collected from the follow-up interviews: Update of any demographic information

8 Development of new cancers (in particular NPC) o Details of the cancer including date of diagnosis presenting symptoms stage of cancer at diagnosis treatment received Development of new co-mobidities other than cancers o Details of the conditions date of diagnosis presenting symptoms treatment Development of new ENT symptoms including o epistaxis o enlarging neck lumps o nose block (unilateral or bilateral) o post-nasal drip o blood-stained nasal discharge of sputum o hearing loss (unilateral or bilateral) Statistical considerations and expected outcomes Detection of NPC cases at earlier stages 20,000 male subjects will be screened for NPC using plasma EBV DNA analysis. In this locality, the incidence of NPC in males aged 40 to 60 is up to 40/100,000 per year. In the previous NPC screening programme involving 1,300 individuals, three NPC cases were identified. These results suggest that the screening programme could potentially advance the diagnosis of NPC, by detecting the cancer before it has progressed to a more advanced stage. In this regard, we estimate that more than 10 NPC cases will be detected during the screening period. The stage distribution of these newly identified NPC cases would be useful for determining the potential beneficial effects of the NPC programme when compared with the stage distribution derived from the historical data. 3 We expected that a much larger proportion of early stage NPC would be detected in the screening study. The proportion of stage I and II NPC cases in the screening study is postulated to be about 90% with a confidence interval between 54% and 99% if we have only 10 NPC cases from the 20,000 screened individuals. However, based on the 1,300 screened subjects with 3 NPC cases in the pilot study, the number of expected NPC cases in the current proposal with 20,000 screened individuals would probably be larger than the conservative estimate of 10 cases. Even if we discount the detection rate by 50%, say we would observe only 1.5 NPC cases instead of 3 per 1300 screened subjects, we would expect to get about 23 NPC cases in the study. Using this estimate, the lower bound of the 95% confidence interval for stage I/II NPC proportion would exceeds 48% (7% stage I plus 41% stage II) if the observed stage I/II NPC proportion is 72% with a 95% CI from 49% to 88%. Therefore, the currently proposed study cohort should be sufficient to detect the down-staging of the NPC cases. Stage distribution of NPC in Hong Kong 3

9 Stage Without screening Rate per 100,000 personyear 5-year survival I 10% 4 92% II 32% 13 80% III 25% 10 73% IV 33% 13 47% Detectable plasma EBV DNA and risk of NPC development In the previous study, we have demonstrated that subjects with detectable EBV DNA at one time point would have higher chance of having detectable plasma EBV DNA in the future. In the currently proposed study, we plan to investigate if individuals with detectable plasma EBV DNA at recruitment would have higher risk of developing NPC in the future. After the first screening session, all individuals with detectable plasma EBV DNA will be subjected to nasal endoscopic examination to confirm or rule out NPC. After this screening period, all participants will be phone interviewed yearly to record if they have developed NPC or other cancers. After this two-year follow-up period, the list of the participants will be matched to the NPC list of the Hong Kong cancer registry yearly to determine if they have developed NPC in that year. With this information, the potential association between the presence of detectable plasma EBV DNA and the risk of NPC development can be assessed. Taking into account the incidence of NPC, we expect that approximately 20 new NPC cases will be identified in the two-year follow-up period and 60 new cases in a 10-year follow-up period. In a previous study, it has been shown that subjects with positive EBV serology would have 4- to 10-fold increased risk of NPC development. 20 The relative risk of NPC development in the subjects with and without detectable EBV DNA at enrolment will be determined. Optimal frequency of screening While the application of plasma EBV DNA analysis in NPC screening appears to be promising, the optimal frequency at which we should screen asymptomatic individuals remains an unanswered question. In the currently proposed study, all participants will be followed up for at least two years after the screening session. If we assume that the screening programme would be able to identify most of the NPC cases, the next screening session would ideally be scheduled before the development and progression of NPC in these screened individual. Therefore, the interval between the first screening session and the first identification of NPC during the follow-up period can be used as a reference for the suitable frequency for the NPC screening programme. Progression-free survival The progression-free survival of the NPC patients identified by the screening programme would be compared with the historical cohort of age- and sex-matched NPC patients in Hong Kong using Kaplan-Meier survival analysis and log-rank test.

10 References 1. Wei WI, Sham JS. Nasopharyngeal carcinoma. Lancet 2005; 365: Lee AW, Sze WM, Au JS et al. Treatment results for nasopharyngeal carcinoma in the modern era: the Hong Kong experience. Int J Radiat Oncol Biol Phys 2005; 61: Leung SF, Zee B, Ma BB et al. Plasma Epstein-Barr viral deoxyribonucleic acid quantitation complements tumor-node-metastasis staging prognostication in nasopharyngeal carcinoma. J Clin Oncol 2006; 24: Yeung WM, Zong YS, Chiu CT et al. Epstein-Barr virus carriage by nasopharyngeal carcinoma in situ. Int J Cancer 1993; 53: Lo YMD, Chan LYS, Lo KW et al. Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res 1999; 59: Lo YMD, Chan LYS, Chan ATC et al. Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999; 59: Lo YMD, Chan ATC, Chan LYS et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res 2000; 60: Chan KCA, Leung SF, Yeung SW, Chan ATC, Lo YMD. Quantitative analysis of the transrenal excretion of circulating EBV DNA in nasopharyngeal carcinoma patients. Clin Cancer Res 2008; 14: Chan KCA, Leung SF, Yeung SW, Chan ATC, Lo YMD. Persistent aberrations in circulating DNA integrity after radiotherapy are associated with poor prognosis in nasopharyngeal carcinoma patients. Clin Cancer Res 2008; 14: Leung SF, Tam JS, Chan ATC et al. Improved accuracy of detection of nasopharyngeal carcinoma by combined application of circulating Epstein-Barr virus DNA and anti-epstein-barr viral capsid antigen IgA antibody. Clin Chem 2004; 50: Leung SF, Lo YMD, Chan ATC et al. Disparity of sensitivities in detection of radiation-naive and postirradiation recurrent nasopharyngeal carcinoma of the undifferentiated type by quantitative analysis of circulating Epstein-Barr virus DNA1,2. Clin Cancer Res 2003; 9: Chan ATC, Lo YMD, Zee B et al. Plasma Epstein-Barr virus DNA and residual disease after radiotherapy for undifferentiated nasopharyngeal carcinoma. J Natl Cancer Inst 2002; 94: Xu J, Wan XB, Huang XF et al. Serologic antienzyme rate of Epstein-Barr virus DNase-specific neutralizing antibody segregates TNM classification in nasopharyngeal carcinoma. J Clin Oncol 2010; 28: Lin JC, Wang WY, Chen KY et al. Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 2004; 350: Lin JC, Wang WY, Liang WM et al. Long-term prognostic effects of plasma epstein-barr virus DNA by minor groove binder-probe real-time quantitative pcr on nasopharyngeal carcinoma patients receiving concurrent chemoradiotherapy. Int J Radiat Oncol Biol Phys 2007; 68:

11 16. Liu Y, Fang Z, Liu L, Yang S, Zhang L. Detection of Epstein-Barr virus DNA in serum or plasma for nasopharyngeal cancer: a meta-analysis. Genet Test Mol Biomarkers 2011; 15: Hou X, Zhao C, Guo Y et al. Different clinical significance of pre- and posttreatment plasma Epstein-Barr virus DNA load in nasopharyngeal carcinoma treated with radiotherapy. Clin Oncol (R Coll Radiol) 2011; 23: Wang WY, Twu CW, Lin WY et al. Plasma Epstein-Barr virus DNA screening followed by (1)F-fluoro-2-deoxy-D-glucose positron emission tomography in detecting posttreatment failures of nasopharyngeal carcinoma. Cancer 2011; 117: Hsu CL, Chang KP, Lin CY et al. Plasma epstein-barr virus DNA concentration and clearance rate as novel prognostic factors for metastatic nasopharyngeal carcinoma. Head Neck 2011; 20. Chien YC, Chen JY, Liu MY et al. Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men. N Engl J Med 2001; 345:

12 Original Research Protocol Title: Prospective 20,000-person nasopharyngeal carcinoma screening programme using plasma Epstein-Barr virus (EBV) DNA analysis Principal investigator: - Professor KC Allen Chan Co-investigators: - Professor YM Dennis Lo - Professor Andrew C van Hasselt - Dr. John KS Woo - Professor Benny Zee - Professor Anthony Chan - Dr. SF Leung Objectives: To investigate if an NPC screening program using plasma EBV DNA would result in the detection of NPC at an earlier stage when compared with the nonscreened population To investigate if subjects with positive plasma EBV DNA would have an increased risk of future NPC development To determine the optimal frequency for NPC screening Background: Nasopharyngeal carcinoma (NPC) is one of the commonest cancers in Southern China and Southeast Asia. 1 In contrast to most other cancers which occur more commonly in elderly subjects, NPC is most prevalent in the middle age. The yearly incidence of NPC in men of age 40 to 60 in Hong Kong is as high as 40 cases per 100, The most important prognostic factor for NPC is the extent of disease involvement at presentation. In patients with localized disease, the 5-year survival probability is up to 90%. 2 This figure drops to below 50% in patients with metastatic disease. In addition, patients with more advanced disease would suffer from a lot more treatment-associated morbidities. 2 Unfortunately, 75% - 90% of NPC patients have already developed local or regional spread at presentation. 2,3 Therefore, screening for early disease in asymptomatic individuals may potentially improve the treatment outcomes of NPC. Epstein-Barr virus infection is an important aetiological factor for NPC. In Asia, EBV genome is detectable in almost all NPC tumour tissues. 4 Making use of the close relationship between EBV infection and NPC, our group has pioneered the development plasma EBV DNA as a tumour marker for NPC in 1999 with the funding support from the Kadoorie Charitable Foundation. 5-9 We showed that EBV DNA is detectable in the plasma of 96% NPC patients using real-time polymerase chain reaction (PCR). In contrast, only 2-7% of individuals without any evidence of NPC would have detectable plasma EBV DNA and at much lower levels than NPC patients. 5,10 The EBV DNA analysis was shown to be much superior to the existing standard surrogate marker for

13 NPC, the EBV IgA serological markers, in terms of sensitivity and specificity for detecting established NPC. 11 For the clinical management of NPC patients, plasma EBV DNA analysis is particularly useful for identifying clinically concealed residual NPC after curative intent radiotherapy 6,12 and for predicting treatment outcomes. 7,13 The prognostic value of circulating EBV DNA analysis has further been shown to be independent of the clinical stage of the NPC patients. 3 The clinical value of circulating EBV DNA analysis has been independently confirmed by other research groups Therefore, it has been proposed that plasma EBV DNA analysis could usefully be incorporated into the staging system of NPC. 3 We further carried out a prospective study to investigate the feasibility of using plasma EBV DNA analysis for the screening of early NPC in high risk asymptomatic individuals from 2007 to Over 1,300 volunteers of age 40 to 60 were recruited for the study. Three plasma EBV DNA analyses were performed for each participant at enrolment, 6 months and 18 months. Serological analysis for EBV viral capsid antigen (VCA) IgA was also performed at enrolment. After each screening session, endoscopic examinations of the nasopharynx were performed within four weeks for all individuals with positive test results (either EBV DNA or EBV VCA-IgA). Another blood sample was collected from each of them on the day of endoscopic examination for plasma EBV DNA testing to investigate the short-term variation of plasma EBV DNA concentrations. Three NPC cases were identified during the screening session at enrolment. All of the three NPC cases were positive for plasma EBV DNA but only one was positive for EBV VCA-IgA indicating that plasma EBV DNA is more sensitive than the serology test for identifying early NPC. In the three patients identified, two of them were completely asymptomatic. The third patient noted a solitary lymph node three months before joining the study. However, fine needle aspiration of the lymph node only showed benign reactive changes but no evidence of malignancy. Remarkably, the tumour of this patient was not detectable during the initial nasal endoscopic examination. Subsequently, prompted by the doubling of the plasma EBV DNA concentration taken two weeks later, a magnetic resonance imaging (MRI) was performed and showed an infiltrating tumour at the nasopharynx. The diagnosis of NPC was then confirmed by repeating the endoscopic examination and taking blind biopsies of the nasopharynx. These results indicate that plasma EBV DNA is useful for the screening of early NPC in apparently healthy individuals. Without this screening programme, these three patients are likely to present with more advanced disease when the symptoms become more overt. In addition to screening for early NPC, this previous study has also provided important information regarding the short- and intermediate-term of variation for plasma EBV DNA in subjects without an NPC. In each of the three screening sessions, 5% to 9% of the participants had detectable levels of EBV DNA in plasma. After a period of two to four weeks, two-thirds of the individuals with initial positive results would become negative for plasma EBV DNA, indicating that the presence of EBV DNA in plasma is only a transient event in a large proportion of subjects without an NPC. On the other hand, EBV DNA is persistently detectable in the plasma of the three NPC patients before treatment. These results suggest that repeating plasma EBV DNA analysis in two weeks for subjects with initial positive results would be useful for cutting down the proportion of subjects requiring nasal endoscopy for confirming or ruling out NPC. This

14 arrangement would significantly enhance the cost-effectiveness of the screening programme. Although the presence of plasma EBV DNA in a large proportion of non- NPC subjects was transient in the short term, subjects with detectable EBV DNA in plasma were significantly more likely to become positive again in the subsequent analyses carried out in 6 months and 18 months. During the previous study, approximately 9% of the individuals had detectable plasma EBV DNA in the two screening sessions carried out in winter time and only around 5% of the subjects had positive results in the session carried out in summer. There was a significant difference in positive rates between the sessions carried out in winter and summer. Using ultracentrifugation analysis, we showed that the plasma EBV DNA in non-cancer subjects are likely to be associated with intact virions instead of existing as free DNA fragments as in the NPC patients. The apparent seasonal variation could be the result from the more active viral replication in winter. If this observation is confirmed with long term studies, it would imply that NPC screening would best be performed in summer to minimize the false-positive rate. Questions to be answered in the current study Although all the three identified patients had early-staged diseases (Stages I and II) in the previous study, it does not have adequate statistical power to show that NPC screening could result in a down-staging of the NPC cases at diagnosis when compared with the non-screened population due to the relatively small size of the cohort. In the currently proposed study, we plan to use a 20,000-person cohort to establish the value of an NPC screening with regard to the down-staging of the identified NPC cases. Furthermore, we aim to determine the optimal frequency for NPC screening by determining the interval between the emergences of new NPC cases after those subjects being screened negative. In the previous study, we have shown that subjects had detectable plasma EBV DNA at one time point were more likely to be positive again subsequently. The biological significance of having recurrently or persistently detectable plasma EBV DNA remains unclear. In this regard, a previous study showed that subjects with a positive EBV serology have a 4- to 10-fold increased risk of developing NPC. 20 As EBV infection is an important aetiological factor for NPC, it is possible that the presence of circulating EBV DNA and EBV antibodies may reflect the pathological processes underlying NPC development. In the currently proposed study, the cohorts of subjects being positive and negative for plasma EBV DNA at recruitment will be followed up to investigate if the presence of detectable EBV DNA in plasma is associated with an increased risk of future NPC development. Study design: Subjects 20,000 Chinese male subjects will be recruited for the module 1 study over a period of two years. This target can be accomplished by recruiting approximately 250 subjects per week (250 subjects/week x 40 weeks/year x 2 years = 20,000 subjects). As the incidence of NPC in male is higher than in females, this study will focus on the male subjects so as to maximize the chance of identifying new NPC cases. Subjects with an existing cancer

15 or a past history of cancer, immunodeficiency or autoimmune diseases will be excluded. Subjects who are on immunosuppressive therapies, e.g., following transplantation, will also be excluded. In the previous proof-of-principle study, approximately 1,300 volunteers were recruited via 12 community visits. Through that exercise, we have established close working relationship with the district councillors and this is essential for the recruitment of volunteers for the currently proposed study. As the public awareness of the prevalence of NPC in Hong Kong is high, the public is generally interested in the voluntary participation in NPC screening programme. In addition to recruiting via community visits with the district councillors, we would invite large corporations in Hong Kong for collaborations for the recruitment of their staff to participate in this NPC screening exercise. Examples of these corporations include the Hospital Authority of Hong Kong, the universities, the banks, the two electricity companies and the public transportation companies. Webpages will be setup for the booking and arrangement of the screening sessions. Plasma EBV DNA analysis Plasma EBV DNA analysis will be performed for each participant during the first visit. An interview by the research nurses will be carried out to document the personal details, and the personal and family medical history relevant to NPC. Quantitative measurement of plasma EBV DNA will be performed by real-time PCR targeting the BamHI-W fragment of the EBV genome as previously described. 5 Plasma EBV DNA analysis will be repeated for all subjects with an initial positive result in two to four weeks. In the previous study, we have demonstrated that NPC patients would have persistently positive results before treatment but two-thirds of the subjects with false-positive results would have undetectable plasma EBV DNA on the follow-up analysis. Therefore, in the currently proposed study, only subjects with persistently detectable plasma EBV DNA will be referred to an otorhinolaryngologist for further assessment. This arrangement could enhance the cost-effectiveness of the screening exercise. Additional imaging study using MRI would be performed as indicated. In our previous studies, VCA-IgA has been shown to have inferior sensitivity over plasma EBV DNA for the detection of NPC, in particular, for early staged disease. 11 In the screening study that we have just completed, all three identified NPC patients had detectable plasma EBV DNA but only one of them was positive for VCA-IgA. The EBV serology test does not appear to provide addition value for the detection of early NPC and, hence, the testing for EBV serology will not be performed for the proposed study. Follow-up After the initial NPC screening session, all the participants will be phoned and interviewed on a yearly basis to update their health records for two to three years. The development of NPC or other cancers will be documented. The interval between NPC development from the time of the plasma EBV DNA screening in patients who subsequently developed NPC in the follow-up period would be useful for determining the

16 optimal frequency of carrying out NPC screening. After the two-year follow-up period, the list of the participants will be matched with the NPC list in the Hong Kong cancer registry yearly to identify new NPC in that year. The follow-up period of these cases is expected to be up to around 10 years. Then, the potential association between the presence of detectable EBV DNA in plasma and the risk of NPC development can also be analyzed. Statistical considerations and expected outcomes Detection of NPC cases at earlier stages In module 1 of this proposed study, 20,000 male subjects will be screened for NPC using plasma EBV DNA analysis. In this locality, the incidence of NPC in males aged 40 to 60 is up to 40/100,000 per year. In the previous NPC screening programme involving 1,300 individuals, three NPC cases were identified. These results suggest that the screening programme could potentially advance the diagnosis of NPC, by detecting the cancer before it has progressed to a more advanced stage. In this regard, we estimate that more than 10 NPC cases will be detected during the screening period. The stage distribution of these newly identified NPC cases would be useful for determining the potential beneficial effects of the NPC programme when compared with the stage distribution derived from the historical data. 3 We expected that a much larger proportion of early stage NPC would be detected in the screening study. The proportion of stage I and II NPC cases in the screening study is postulated to be about 90% with a confidence interval between 54% and 99% if we have only 10 NPC cases from the 20,000 screened individuals. However, based on the 1,300 screened subjects with 3 NPC cases in the pilot study, the number of expected NPC cases in the current proposal with 20,000 screened individuals would probably be larger than the conservative estimate of 10 cases. Even if we discount the detection rate by 50%, say we would observe only 1.5 NPC cases instead of 3 per 1300 screened subjects, we would expect to get about 23 NPC cases in the study. Using this estimate, the lower bound of the 95% confidence interval for stage I/II NPC proportion would exceeds 48% (7% stage I plus 41% stage II) if the observed stage I/II NPC proportion is 72% with a 95% CI from 49% to 88%. Therefore, the currently proposed study cohort should be sufficient to detect the down-staging of the NPC cases. Stage distribution of NPC in Hong Kong 3 Rate per 100,000 personyear Stage Without screening 5-year survival I 10% 4 92% II 32% 13 80% III 25% 10 73% IV 33% 13 47% Detectable plasma EBV DNA and risk of NPC development In the previous study, we have demonstrated that subjects with detectable EBV DNA at one time point would have higher chance of having detectable plasma EBV DNA in the future. In the currently proposed study, we plan to investigate if individuals with detectable plasma EBV DNA at recruitment would have higher risk of developing NPC

17 in the future. After the first screening session, all individuals with detectable plasma EBV DNA will be subjected to nasal endoscopic examination to confirm or rule out NPC. After this screening period, all participants will be phone interviewed yearly to record if they have developed NPC or other cancers. After this two-year follow-up period, the list of the participants will be matched to the NPC list of the Hong Kong cancer registry yearly to determine if they have developed NPC in that year. With this information, the potential association between the presence of detectable plasma EBV DNA and the risk of NPC development can be assessed. Taking into account the incidence of NPC, we expect that approximately 20 new NPC cases will be identified in the two-year follow-up period and 60 new cases in a 10-year follow-up period. In a previous study, it has been shown that subjects with positive EBV serology would have 4- to 10-fold increased risk of NPC development. 20 The relative risk of NPC development in the subjects with and without detectable EBV DNA at enrolment will be determined. Optimal frequency of screening While the application of plasma EBV DNA analysis in NPC screening appears to be promising, the optimal frequency at which we should screen asymptomatic individuals remains an unanswered question. In the currently proposed study, all participants will be followed up for at least two years after the screening session. If we assume that the screening programme would be able to identify most of the NPC cases, the next screening session would ideally be scheduled before the development and progression of NPC in these screened individual. Therefore, the interval between the first screening session and the first identification of NPC during the follow-up period can be used as a reference for the suitable frequency for the NPC screening programme.

18 References 1. Wei WI, Sham JS. Nasopharyngeal carcinoma. Lancet 2005; 365: Lee AW, Sze WM, Au JS et al. Treatment results for nasopharyngeal carcinoma in the modern era: the Hong Kong experience. Int J Radiat Oncol Biol Phys 2005; 61: Leung SF, Zee B, Ma BB et al. Plasma Epstein-Barr viral deoxyribonucleic acid quantitation complements tumor-node-metastasis staging prognostication in nasopharyngeal carcinoma. J Clin Oncol 2006; 24: Yeung WM, Zong YS, Chiu CT et al. Epstein-Barr virus carriage by nasopharyngeal carcinoma in situ. Int J Cancer 1993; 53: Lo YMD, Chan LYS, Lo KW et al. Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res 1999; 59: Lo YMD, Chan LYS, Chan ATC et al. Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999; 59: Lo YMD, Chan ATC, Chan LYS et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res 2000; 60: Chan KCA, Leung SF, Yeung SW, Chan ATC, Lo YMD. Quantitative analysis of the transrenal excretion of circulating EBV DNA in nasopharyngeal carcinoma patients. Clin Cancer Res 2008; 14: Chan KCA, Leung SF, Yeung SW, Chan ATC, Lo YMD. Persistent aberrations in circulating DNA integrity after radiotherapy are associated with poor prognosis in nasopharyngeal carcinoma patients. Clin Cancer Res 2008; 14: Leung SF, Tam JS, Chan ATC et al. Improved accuracy of detection of nasopharyngeal carcinoma by combined application of circulating Epstein-Barr virus DNA and anti-epstein-barr viral capsid antigen IgA antibody. Clin Chem 2004; 50: Leung SF, Lo YMD, Chan ATC et al. Disparity of sensitivities in detection of radiation-naive and postirradiation recurrent nasopharyngeal carcinoma of the undifferentiated type by quantitative analysis of circulating Epstein-Barr virus DNA1,2. Clin Cancer Res 2003; 9: Chan ATC, Lo YMD, Zee B et al. Plasma Epstein-Barr virus DNA and residual disease after radiotherapy for undifferentiated nasopharyngeal carcinoma. J Natl Cancer Inst 2002; 94: Xu J, Wan XB, Huang XF et al. Serologic antienzyme rate of Epstein-Barr virus DNase-specific neutralizing antibody segregates TNM classification in nasopharyngeal carcinoma. J Clin Oncol 2010; 28: Lin JC, Wang WY, Chen KY et al. Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 2004; 350: Lin JC, Wang WY, Liang WM et al. Long-term prognostic effects of plasma epstein-barr virus DNA by minor groove binder-probe real-time quantitative pcr on nasopharyngeal carcinoma patients receiving concurrent chemoradiotherapy. Int J Radiat Oncol Biol Phys 2007; 68:

19 16. Liu Y, Fang Z, Liu L, Yang S, Zhang L. Detection of Epstein-Barr virus DNA in serum or plasma for nasopharyngeal cancer: a meta-analysis. Genet Test Mol Biomarkers 2011; 15: Hou X, Zhao C, Guo Y et al. Different clinical significance of pre- and posttreatment plasma Epstein-Barr virus DNA load in nasopharyngeal carcinoma treated with radiotherapy. Clin Oncol (R Coll Radiol) 2011; 23: Wang WY, Twu CW, Lin WY et al. Plasma Epstein-Barr virus DNA screening followed by (1)F-fluoro-2-deoxy-D-glucose positron emission tomography in detecting posttreatment failures of nasopharyngeal carcinoma. Cancer 2011; 117: Hsu CL, Chang KP, Lin CY et al. Plasma epstein-barr virus DNA concentration and clearance rate as novel prognostic factors for metastatic nasopharyngeal carcinoma. Head Neck 2011; 20. Chien YC, Chen JY, Liu MY et al. Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men. N Engl J Med 2001; 345:

20 Summary of changes between Original and Final Research Protocol The only difference in the original and the final research protocol is that participants with persistently positive plasma EBV DNA would be investigated by nasal endoscopy, and MRI would be performed only as clinically indicated, in the original protocol. In the final research protocol, all screened positive participants with persistently positive plasma EBV DNA would be investigated by both nasal endoscopy and MRI.

21 Final statistical plan After the screening of all the participants with plasma EBV DNA analysis, the number and percentage of subjects with transiently and persistently detectable plasma EBV DNA would be determined. For the subjects who were subsequently confirmed of having NPC, their stage distribution would be compared with the stage distribution of NPC recorded in the latest Hong Kong Cancer Registry ( The progression-free survival of the NPC patients identified by the study would be compared with a cohort of sex- and age-matched NPC patients in a historical cohort comprising of all patients treated in all oncology centers in Hong Kong (Lee et al. Int J Radiat Oncol Biol Phys 2005;61: ). The demographics of the two cohorts would be compared.

22 Original statistical plan After the screening of all the participants with plasma EBV DNA analysis, the number and percentage of subjects with transiently and persistently detectable plasma EBV DNA would be determined. For the subjects who were subsequently confirmed of having NPC, their stage distribution would be compared with the stage distribution of NPC recorded in the latest Hong Kong Cancer Registry ( The predicted 5-year survival of the NPC patients identified by this screening program would be estimated based on the stage distribution of the patient and the stage-specific survival from previous studies (Lee et al. Int J Radiat Oncol Biol Phys 2005;61: ).

23 Summary of changes the final and the original statistical plan The only difference between the original and final statistical plan is the inclusion of the comparison of the actual progression-free survival between the NPC patients identified by our screening program and a sex- and age-matched historical cohort is included in the final statistical analysis. In the original statistical plan, the 5-year progression-free survival of the NPC patients identified by this screening program would be estimated based on their stage distribution and the reported stage-specific survival of a previous cohort (Lee et al. Int J Radiat Oncol Biol Phys 2005;61: ).